One approach to improve the efficacy of in vivo gene therapy, with the aim at enhancing expression of a transgene, involves utilization of ultrasound to facilitate transfection of DNA into cells. Manome et al., Circulation, 99(20):2617–20(1999), incorporated herein by reference, disclose transfection of naked plasmid DNA into colon carcinoma cells in vitro and in vivo by using ultrasound. In this study, a reporter pcDNA3-lacZ plasmid, containing Escherichia coli lacZ or the beta-galactosidase gene (beta-gal), and a neomycin 3′-phosphotransferase gene (neo) has been used for evaluation of transfer efficiency. MC38 cells, murine colon carcinoma cells, have been implanted in syngeneic mice and plasmid with naked DNA injected in the mice and the cells. The cells were subjected to continuous ultrasound exposure at 1.0-MHz, 20 W/cm2. In a transient assay, significant numbers of cells were transduced with the beta-galactosidase gene.
Lawrie et al., Circulation, 99(20):2617–20(1999), reported that ultrasound enhances gene expression after transfection of vascular cells in vitro. Lawrie et al. investigated the use of adjunctive ultrasound to enhance nonviral gene delivery. In this study, cultured porcine vascular smooth muscle cells and endothelial cells were transfected with naked or liposome-complexed luciferase reporter plasmid. The luciferase activity after lipofection of endothelial cells was enhanced by adjunctive ultrasound exposure. Ultrasound exposure had no effect on cell viability, although it inhibited vascular smooth muscle cells, but not endothelial cell proliferation.
Shohet et al., Circulation, 101(22):2554–6(2000), incorporated herein by reference, describe a noninvasive method for transduction of a gene. In this study, recombinant adenovirus containing beta-gaactosidase and driven by a constitutive promoter was attached to the surface of albumin-coated, perfluoropropane-filled microbubbles. Then, microbubbles were infused into the jugular vein of rats with or without simultaneous echocardiography to effectively deliver the adenovirus to rat myocardium. The hearts of all rats that underwent ultrasound-mediated destruction of microbubbles containing virus showed myocardial expression of the beta-galactosidase transgene.
Huber et al, Gene Ther. 7(17):1516–25(2000), reported that gene therapy, as a form of molecular medicine, is expected to have a major impact on medical treatments in the future, but that the clinical use of gene therapy is hampered by inadequate gene delivering systems to ensure sufficient, accurate and safe DNA uptake in the target cells in vivo. Huber et al. reported that nonviral transfection methods might have the advantage of safe application, but it would be helpful to increase their transfection rates, especially in vivo. Huber's studies focused on using ultrasound to provide an enhanced transfer of DNA plasmids in vitro and in vivo. In vitro, the beta-galactosidase and luciferase DNA reporter plasmid were transfected into four cell lines (NIH 3T3 fibroblasts, malignant melanoma Mewo, HeLa, Dunning prostate tumor R3327-AT1). Ultrasound induced a 55-(Mewo) to 220-fold (AT1) stimulation resulting in transfection efficiencies in vitro between 2% (Mewo) and 12% (AT1). The in vivo stimulation was assessed in the Dunning prostate tumor R3327-AT1 implanted subcutaneously in Copenhagen rats using the beta-galactosidase reporter. After intratumoral DNA injection, focused ultrasound induced a 10-fold increase of beta-galactosidase positive cells in histology and a 15-fold increase of beta-galactosidase protein expression in the ELISA assay. In contrast, ultrasound was not found to enhance reporter gene expression after intravenous plasmid application.
In another study, Anwer et al., Gene Ther. 7(21):1833–9 (2000), investigated the impact of a localized application of ultrasound on gene transfer to primary tumors following systemic administration of cationic lipid based transfection complexes. Anwer et al. reported that it had been previously shown that systemic administration of (N-[(1-(2-3-dioleyloxy)propyl)]-N—N—N-trimethylammonium chloride):cholesterol-based transfection complexes to tumor-bearing mice resulted in expression in the tumor and other tissues, primarily the lungs.
A microbubble-enhanced ultrasound technique for vascular gene delivery was reported by Lawrie et al., Gene Ther.; 7(23):2023–2027 (2000), incorporated herein by reference. Lawrie noted that progress in cardiovascular gene therapy has been hampered by concerns over the safety and practicality of viral vectors and the inefficiency of current nonviral transfection techniques.
U.S. Pat. Re. 36,939 to Tachibana et al. describes a booster comprising microbubbles of a gas in a liquid, e.g. about 4×107 cells/ml of microbubbles of a gas having a diameter of 0.1 to 100 um in 3 to 5% human serum albumin solution, and a pharmaceutical liquid composition comprising the booster and a medicament, which are useful for the therapy of various diseases together with exposure to ultrasonic waves.
U.S. Pat. No. 5,542,935 to Unger, et al., discloses a therapeutic delivery system comprising gaseous precursor-filled liposomes having encapsulated therein a contrast agent or drug.
PCT publication W08902464 discloses a method of introducing material into living mammalian cells, or of fusing material with the cells. The method comprises subjecting the cells in liquid suspension in the presence of the material to ultrasonic excitation sufficient to traumatize the cells. The material introduced into the cells, or into a cell membrane, is preferably DNA or RNA or a protein.
China Patent No. 1056124 discloses a gene conduction method using ultrasonic waves for genetic engineering of both animals and plants. A biological material in DNA solution treated with an ultrasonic wave causes the cell membranes of the biological material to change in structure such that DNA molecules in the solution may diffuse into the cells.
PCT publication W09806864 discloses methods for using local heat to control gene expression. The heat shock protein (hsp) gene promoter is recombined with a selected therapeutic gene and expressed in selected cells. Local controlled heating is used to activate the hsp promoter, for example by using focused ultrasound.
PCT publication W00042988 discloses a method of identifying and/or treating tissue having leukocytes adhered thereto which utilizes a material that selectively attaches to leukocytes adhered to a patient's tissue. The material may be a gas-filled microbubble contrast agent that selectively attaches to activated inflamed tissue. The microbubbles attached to activated leukocytes may be located by ultrasound echography, and inflamed tissue may be treated by a drug or gene sequence carried by the microbubble contrast agent.
U.S. Pat. No. 6,265,387 BI and PCT application WO 00/50617, published Aug. 31, 2000 disclose methods for gene therapy which involve the delivery of a polynucleotide to a cell in a mammal by injecting the polynucleotide into a blood vessel connected to the cell such that the polynucleotide is transfected into the cell and expressed to therapeutic levels. In these disclosures, this intravascular route of administration is increased by increasing the permeability of the tissues blood vessel by increasing the intravascular hydrostatic (physical) pressure or increasing the osmotic pressure.
According to the present invention, gene expression in the liver is accomplished by injecting DNA or equivalent into the blood peripherally and isonifying the liver with ultrasound. The ultrasound waves alone, when enhanced by disrupted microspheres, optionally with increased injection pressure, will result in efficient transfection of DNA or equivalent into the nucleus.